11-04-11 Proteomics The science of proteomics Applications of proteomics Proteomic methods

1. 11-04-11 Proteomics The science of proteomics Applications of proteomics Proteomic methods a. protein purification b. protein sequencing c. mass spectrometry

2. The proteome is the entire set of proteins expressed by a genome, cell, tissue or organism More specifically, it is the set of expressed proteins in a given type of cells or an organism at a given time under defined conditions 1. The cellular proteome - the collection of proteins in a particular cell type under a particular set of conditions, eg. exposure to hormone stimulation 2. The complete proteome - complete set of proteins from all of the various cellular proteomes 3. Subsets: the mitochondrial proteome, the viral proteome, etc.

3. The bacterial proteome is roughly equivalent to the bacterial genome; eukaryotic proteomes are much larger than eukaryotic genomes 1. Alternative splicing 2. Protein modification In addition to the sequence information in proteins the proteome can also include 1. Protein folding 2. Functional interactions between proteins and other molecules

4. Tools for proteomic research 1. Polyacrylamide gel electrophoresis, to separate proteins a. SDS gel electrophoresis separates proteins by mass b. Isoelectric focusing separates proteins by charge c. Two-dimensional gel electrophoresis (2D PAGE) d. Special methods, eg gel electrophoresis in the presence of urea, low pH and Triton X-100 2. Cell fractionation 3. Various methods for protein purification 4. Protein sequencing 5. Mass spectrometry of peptides and intact proteins 6. Yeast two-cell hybrid system - protein-protein interactions

5. Applications of proteomics 1. Comparison of protein sequences to establish similarities that define protein families 2. Comparison of the same protein in different species to reveal evolutionary relationships 3. Search and discovery of common motifs in proteins that define functions, destinations or processing 4. Sequence data allow a molecular understanding of diseases 5. Maps of protein interactions help define critical steps in cellular metabolism • Proteomic data can inform treatment of disease

6. Cell Fractionation Differential centrifugation permits separation of different cellular components following cell disruption Cell disruption yields a homogenate Centrifugation of the homogenate yields fractions called the pellet and supernatant Differential centrifugation:The supernatant may again be centrifuged at higher speed to yield another pellet. The fraction used is called the crude extract

8. Subsequent purification of a specific protein fraction requires a specific assay 1. Enzyme assays are usually quick and easy 2. The degree of purification is followed by the specific activity (specific protein/total protein) NADH absorbs light at 340 nm

9. Salting in (or out) exploits the solubility of a protein in different salt concentrations

10. Dialysis allows removal (or addition) of small molecules that are permeable to the dialysis membrane

11. Exclusion chromatography separates proteins according to their molecular weights

12. Ion exchange chromatography separates proteins according to their molecular charge

13. Affinity chromatography separates proteins according to their affinity for specific ligands

14. High Pressure Liquid Chromatography (HPLC) A high pressure version of liquid chromatography providing greater speed and resolution The elution of proteins from chromatography columns is often monitored by UV light absorbance

15. SDS gel electrophoresis depends on the ability of the anion detergent sodium dodecyl sulfate to dissolve proteins SDS coats the protein, causing it to be denatured to a uniform ellipsoidal shape that is coated with a uniform negative charge called a micelle Proteins dissolved in SDS exist as monomers that can then be separated by electrophoresis through a polyacrylamide matrix that serves to sieve the proteins according to their molecular weights

16. Small proteins migrate more rapidly Large proteins migrate more slowly

17. Following electrophoresis the gel is removed from the apparatus and stained to reveal the proteins If the protein samples are run in parallel with proteins of known molecular weight the molecular size of the unknown proteins can be estimated

18. Isoelectric focusing separates proteins according to their isoelectric point

19. Two-dimensional polyacrylamide electrophoresis (2D PAGE) provides very high resolution of complex protein mixtures

21. SDS gel electrophoesis is often used to follow the progress of a protein purification

23. The amino acid composition of a protein can be determined by complete acid hydrolysis For example: Ala-Gly-Asp-Phe-Arg-Gly The protein is hyrdolyzed to free amino acids, which are then reacted with fluorescamine

25. Edman Degradation is used to determine the primary structure of a protein

26. The number of amino acids that can be determined by Edman Degradation is limited to about 50 amino acids. Larger proteins are enzymatically or chemically broken into fragments and sequenced individually

29. Protein-protein interactions can be detected using the yeast two-hybrid system http://en.wikipedia.org/wiki/Two-hybrid_screening